A novel lectin with a distinct Gal_Lectin and CUB domain mediates haemocyte phagocytosis in oyster Crassostrea gigas.

Invertebrate lectins exhibit structural diversity and play crucial roles in the innate immune responses by recognizing and eliminating pathogens. In the present study, a novel lectin containing a Gal_Lectin, a CUB and a transmembrane domain was identified from the Pacific oyster Crassostrea gigas (defined as CgGal-CUB). CgGal-CUB mRNA was detectable in all the examined tissues with the highest expression in adductor muscle (11.00-fold of that in haemocytes, p < 0.05). The expression level of CgGal-CUB mRNA in haemocytes was significantly up-regulated at 3, 24, 48 and 72 h (8.37-fold, 12.13-fold, 4.28-fold and 10.14-fold of that in the control group, respectively) after Vibrio splendidus stimulation. The recombinant CgGal-CUB (rCgGal-CUB) displayed binding capability to Mannan (MAN), peptidoglycan (PGN), D-(+)-Galactose and L-Rhamnose monohydrate, as well as Gram-negative bacteria (Escherichia coli, V. splendidus and Vibrio anguillarum), Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, and Bacillus sybtilis) and fungus (Pichia pastoris). rCgGal-CUB was also able to agglutinate V. splendidus, and inhibit V. splendidus growth. Furthermore, rCgGal-CUB exhibited the activities of enhancing the haemocyte phagocytosis towards V. splendidus, and the phagocytosis rate of haemocytes was descended in blockage assay with CgGal-CUB antibody. These results suggested that CgGal-CUB served as a pattern recognition receptor to bind various PAMPs and bacteria, and enhanced the haemocyte phagocytosis towards V. splendidus.

Genome-wide identification of the HSP70 genes in Pacific oyster Magallana gigas and their response to heat stress.

Heat shock protein 70 (HSP70), the most prominent and well-characterized stress protein in animals, plays an important role in assisting animals in responding to various adverse conditions. In the present study, a total of 113 HSP70 gene family members were identified in the updated genome of Magallana gigas (designated MgHSP70) (previously known as Crassostrea gigas). There were 75, 12, 11, and 8 HSP70s located in the cytoplasm, nucleus, mitochondria, and endoplasmic reticulum, respectively, and 7 HSP70s were located in both the nucleus and cytoplasm. Among 113 MgHSP70 genes, 107 were unevenly distributed in 8 chromosomes of M. gigas with the greatest number in chromosome 07 (61 genes, 57.01%). The MgHSP70 gene family members were mainly assigned into five clusters, among which the HSPa12 subfamily underwent lineage-specific expansion, consisting of 89 members. A total of 68 MgHSP70 genes (60.18%) were tandemly duplicated and formed 30 gene pairs, among which 14 gene pairs were under strong positive selection. In general, the expression of MgHSP70s was tissue-specific, with the highest expression in labial palp and gill and the lowest expression in adductor muscle and hemocytes. There were 35, 31, and 47 significantly upregulated genes at 6, 12, and 24 h after heat shock treatment (28 °C), respectively. The expression patterns of different tandemly duplicated genes exhibited distinct characteristics after shock treatment, indicating that these genes may have different functions. Nevertheless, genes within the same tandemly duplicated group exhibit similar expression patterns. Most of the tandemly duplicated HSP70 gene pairs showed the highest expression levels at 24 h. This study provides a comprehensive description of the MgHSP70 gene family in M. gigas and offers valuable insights into the functions of HSP70 in the mollusc adaptation of oysters to environmental stress.

A novel surface marker CD49d promotes TNF expression in oyster agranulocytes by mediating the MAPK pathway.

CD49d, encoded by the gene Integrin α4, is a significant member of cell adhesion receptors, which is widely expressed in various immune cells to trigger immune responses against invading pathogens. In the present study, the expression of CgCD49d and its regulatory role in TNF expression were investigated in the Pacific oyster Crassostrea gigas. There were five Int-alpha domains, an Integrin_alpha2 region and a unique FG-GAP repeat region inserted identified in CgCD49d. CgCD49d transcript was specifically expressed in haemocytes, and its mRNA expression level in haemocytes increased after LPS and Vibrio splendidus stimulation. After CgCD49d was blocked by using its antibody, the phosphorylation level of CgJNK in the MAPK signaling pathway and CgTNF transcripts decreased significantly post V. splendidus stimulation. After phosphorylation level of CgJNK was inhibited by using its inhibitor, the nuclear translocation of CgRel was restrained and CgTNF transcripts also decreased significantly post V. splendidus stimulation. Furthermore, CgCD49d was found to be mainly expressed in the agranulocyte subpopulation, and Alexa Fluor 488-conjugated CgCD49d antibody labeled agranulocytes with a circle of green fluorescence signals on CgCD49d+ agranulocyte surface under Confocal microscopy, which accounted for 24.9 ± 4.53% of total haemocytes. Collectively, these results suggested that CgCD49d promoted TNF expression in oyster haemocytes against bacterial invasion by mediating MAPK pathway, and it could be used as a surface marker to type and sort a subset of agranulocyte subpopulation among haemocytes.

Norepinephrine regulates TNF expression via the A1AR-p38 MAPK-Relish pathway in granulocytes of oyster Crassostrea gigas.

Norepinephrine (NE) is involved in regulating cytokine expression and phagocytosis of immune cells in the innate immunity of vertebrates. In the present study, the modulation mechanism of NE on the biosynthesis of TNFs in oyster granulocytes was explored. The transcripts of CgTNF-1, CgTNF-2 and CgTNF-3 were highly expressed in granulocytes, and they were significantly up-regulated after LPS stimulation, while down-regulated after NE treatment. The phagocytic rate and apoptosis index of oyster granulocytes were also triggered by LPS stimulation and suppressed by NE treatment. The mRNA expressions of CgMAPK14 and CgRelish were significantly induced after NE treatment, and the translocation of CgRelish from cytoplasm to nucleus was observed. The concentration of intracellular Ca2+ in granulocytes was significantly up-regulated upon NE incubation, and this trend reverted after the treatment with DOX (specific antagonist for NE receptor, CgA1AR-1). No obvious significance was observed in intracellular cAMP concentrations in the PBS, NE and NE + DOX groups. Once CgA1AR-1 was blocked by DOX, the mRNA expressions of CgMAPK14 and CgRelish were significantly inhibited, and the translocation of CgRelish from cytoplasm to nucleus was also dramatically suppressed, while the mRNA expression of CgTNF-1 and the apoptosis index increased significantly to the same level with those in LPS group, respectively. These results collectively suggested that NE modulated TNF expression in oyster granulocyte through A1AR-p38 MAPK-Relish signaling pathway.

Involvement of metabotropic glutamate receptors in regulation of immune response in the Pacific oyster Crassostrea gigas.

Metabotropic glutamate receptors (mGluRs) play a pivotal role in the neuroendocrine-immune regulation. In this study, eight mGluRs were identified in the Pacific Oyster Crassostrea gigas, which were classified into three subfamilies based on genetic similarity. All CgmGluRs harbor variable numbers of PBP1 domains at the N-terminus. The sequence and structural features of CgmGluRs are highly similar to mGluRs in other species. A uniformly upregulated expression of CgmGluRs was observed during D-shaped larval stage compared to early D-shaped larval stage. The transcripts of CgmGluRs were detectable in various tissues of oyster. Different CgmGluR exhibited diverse expression patterns response against different PAMP stimulations, among which CgmGluR5 was significantly downregulated under these stimulations, reflecting its sensitivity and broad-spectrum responsiveness to microbes. Following LPS stimulation, the mRNA expression of CgmGluR5 and CgCALM1 in haemocytes was suppressed within 6 h and returned to normal levels by 12 h. Inhibition of CgmGluR5 activity resulted in a significant reduction in CgCALM1 expression after 12 h. Further KEGG enrichment analysis suggested that CgmGluR5 might modulate calcium ion homeostasis and metabolic pathways by regulating CgCALM1. This research delivers the systematic analysis of mGluR in the Pacific Oyster, offering insights into evolutionary characteristics and immunoregulatory function of mGluR in mollusks.

The c.503A>G polymorphism in ZIP1-II of Pacific oyster Crassostrea gigas associated with zinc content.

The Pacific oyster Crassostrea gigas is renowned for its high zinc content, but the significant variation among individuals diminishes its value as a reliable source of zinc supplementation. The Zrt/Irt-like protein 1 (ZIP1), a pivotal zinc transporter that facilitates zinc uptake in various organisms, plays crucial roles in regulating zinc content. In the present study, polymorphisms of a ZIP1 gene in C. gigas (CgZIP1-II) were investigated, and their association with zinc content was evaluated through preliminary association analysis in 41 oysters and verification analysis in another 200 oysters. A total of 17 single nucleotide polymorphisms (SNPs) were identified in the exonic region of CgZIP1-II gene, with c.503A>G significantly associated with zinc content. Protein sequence and structure prediction showed that c.503A>G caused a p.Met110Val nonsynonymous mutation located in the metal-binding region of CgZIP1-II, which could influence its affinity for zinc ions, thereby modulating its zinc transport functionality. These results indicate the potential influence of CgZIP1-II polymorphisms on zinc content and provide candidate markers for selecting C. gigas with high zinc content.

CgADAR1 involved in regulating the synthesis of interferon-like protein in Crassostrea gigas.

Adenosine deaminases acting on RNA 1 (ADAR1) is a dsRNA adenosine (A)-to-inosine (I) editing enzyme that regulates the innate immune response against virus invasion. In the present study, a novel CgADAR1 was identified from the oyster Crassostrea gigas. The open reading frame (ORF) of CgADAR1 was of 3444 bp encoding a peptide of 1147 amino acid residues with two Zα domains, one dsRNA binding motif (DSRM) and one RNA adenosine deaminase domain (ADEAMc). The mRNA transcripts of CgADAR1 were detected in all the examined tissues, with higher expression levels in mantle and gill, which were 7.11-fold and 4.90-fold (p < 0.05) of that in labial palp, respectively. The mRNA transcripts of CgADAR1 in haemocytes were significantly induced at 24 h and 36 h after Poly (A: U) stimulation, which were 6.03-fold (p < 0.01) and 1.37-fold (p < 0.001) of that in control group, respectively. At 48 h after Poly (A:U) stimulation, the mRNA expression of CgRIG-Ⅰ, CgIRF8 and CgIFNLP significantly increased, which were 4.36-fold (p < 0.001), 1.82-fold (p < 0.05) and 1.92-fold (p < 0.05) of that in control group. After CgADAR1 expression was inhibited by RNA interference (RNAi), the mRNA expression levels of CgMDA5, CgRIG-Ⅰ, CgTBK1, CgIRF8 and CgIFNLP were significantly increased, which were 11.88-fold, 11.51-fold, 2.22-fold, 2.85-fold and 2.52-fold of that in control group (p < 0.001), and the phosphorylation level of CgTBK1 was also significantly increased. These results suggested that CgADAR1 played a regulation role in the early stages of viral infection by inhibiting the synthesis of interferon-like protein.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

Polymorphisms in the cysteine dioxygenase gene and their association with taurine content in the Pacific oyster Crassostrea gigas.

The Pacific oyster Crassostrea gigas is rich in taurine, which is crucial for its adaptation to the fluctuating intertidal environment and presents significant potential in improving taurine nutrition and boosting immunity in humans. Cysteine dioxygenase (CDO) is a key enzyme involved in the initial step of taurine biosynthesis and plays a crucial role in regulating taurine content in the body. In the present study, polymorphisms of CDO gene in C. gigas (CgCDO) and their association with taurine content were evaluated in 198 individuals. A total of 24 single nucleotide polymorphism (SNP) loci were identified in the exonic region of CgCDO gene by direct sequencing. Among these SNPs, c.279G>A and c.287C>A were found to be significantly associated with taurine content, with the GG and AA genotype at the two loci exhibiting enhanced taurine accumulation (p < 0.05). Haplotype analysis revealed that the 279GG/287AA haplotype had the highest taurine content of 29.24 mg/g, while the 279AA/287CC haplotype showed the lowest taurine content of 21.19 mg/g. These results indicated that the SNPs of CgCDO gene could influence the taurine content in C. gigas and have potential applications in the selective breeding of high-taurine varieties.

The expression patterns of exosomal miRNAs in the Pacific oyster after high-temperature stress or Vibrio stimulation.

The exosomal miRNA plays a crucial role in the intercellular communication response to environmental stress and pathogenic stimulation. In the present study, the expression of exosomal miRNAs in the Pacific oyster Crassostrea gigas after high-temperature stress or Vibrio splendidus stimulation was investigated through high-throughput sequencing. The exosomes were identified to be teardrop-like vesicles with the average size of 81.7 nm by transmission electron microscopy. There were 66 known miRNAs and 33 novel miRNAs identified, of which 10 miRNAs were differentially expressed after both high-temperature stress and Vibrio stimulation compared to the control group. A total of 1868 genes were predicted as the putative targets of miRNAs, of which threonine aspartase 1-like was targeted by the highest number of related miRNAs. The robustness and reliability of miRNA expression from the sRNA sequencing data were verified by employing eight miRNAs for qPCR. GO and KEGG clustering analyses revealed that apoptosis was significantly enriched by the target genes of differentially expressed exosomal miRNAs after high-temperature stress, and autophagy and cytokine activity were significantly enriched after Vibrio stimulation. Energy metabolism was found to be significantly shared in the target gene enrichments after both high-temperature stress and Vibrio stimulation. These findings would improve our understanding of the regulatory mechanisms of exosomal miRNAs in C. gigas after high-temperature stress or Vibrio stimulation.

A trace amine associated receptor mediates antimicrobial immune response in the oyster Crassostrea gigas.

Trace amine-associated receptors (TAARs) are a class of G protein-coupled receptors, playing an immunomodulatory function in the neuroinflammatory responses. In the present study, a TAAR homologue with a 7tm_classA_rhodopsin-like domain (designated as CgTAAR1L) was identified in oyster Crassostrea gigas. The abundant CgTAAR1L transcripts were detected in visceral ganglia and haemocytes compared to other tissues, which were 55.35-fold and 32.95-fold (p < 0.01) of those in adductor muscle, respectively. The mRNA expression level of CgTAAR1L in haemocytes significantly increased and reached the peak level at 3 h after LPS or Poly (I:C) stimulation, which was 4.55-fold and 12.35-fold of that in control group, respectively (p < 0.01). After the expression of CgTAAR1L was inhibited by the injection of its targeted siRNA, the mRNA expression levels of interleukin17s (CgIL17-1, CgIL17-5 and CgIL17-6), and defensin (Cgdefh1) significantly decreased at 3 h after LPS stimulation, which was 0.51-fold (p < 0.001), 0.39-fold (p < 0.01), 0.48-fold (p < 0.05) and 0.41-fold (p < 0.05) of that in the control group, respectively. The nuclear translocation of Cgp65 protein was suppressed in the CgTAAR1L-RNAi oysters. Furthermore, the number of Vibrio splendidus in the haemolymph of CgTAAR1L-RNAi oysters significantly increased (4.11-fold, p < 0.001) compared with that in the control group. In contrast, there was no significant difference in phagocytic rate of haemocytes to V. splendidus in the CgTAAR1L-RNAi oysters. These results indicated that CgTAAR1L played an important role in the immune defense against bacterial infection by inducing the expressions of interleukin and defensin.

An LPS-induced TNF-α factor involved in immune response of oyster Crassostrea gigas by regulating haemocytes apoptosis.

LPS induced TNF-α Factor (LITAF) is a transcription factor widely involving in activation of Tumor Necrosis Factor (TNF) and other cytokines in the inflammatory response. In the present study, a homologue of LITAF with a conserved LITAF domain was identified from the Pacific oyster Crassostrea gigas. The transcripts of CgLITAF were detected in all examined tissues with highest expression in hepatopancrease. The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgLITAF protein in haemocytes. While the mRNA level of CgLITAF changed slightly after LPS stimulation. When the siRNA of CgLITAF was injected to inhibit its expression, the apoptotic level of haemocytes decreased observably after LPS stimulation. Consistently, the transcripts of CgTNF3 and CgTNF4 (LOC105343080, LOC105341146), the apoptotic-related molecules including CgBax, CgCytochrome c, CgCaspase9 and CgCaspase3, were significantly suppressed in the CgLITAF-RNAi oysters. While the mRNA expression level of CgBcl was enhanced significantly in the CgLITAF-RNAi oysters. These results indicated that CgLITAF promoted haemocyte apoptosis by regulating the expression of apoptotic-related factors, suggesting its important role in the immune response of oysters.

CgPHB2 involved in the haemocyte mitophagy in response to Vibrio splendidus stimulation in Pacific oyster Crassostrea gigas.

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of CgPHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. CgPHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of CgPHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of CgPHB2 protein was also enhanced at 12-24 h compared to control group. Furthermore, the green signals of CgPHB2 were colocalized respectively with the red signals of mitochondria and CgLC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of CgPHB2 and mtphagy Dye was significantly increased. The direct interaction between CgPHB2 and CgLC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that CgPHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters.

The involvement of interferon regulatory factor 8 in regulating the proliferation of haemocytes in oyster Crassostrea gigas.

Interferon regulatory factor 8 (IRF8) is an important transcriptional regulatory factor involving in multiple biological process, such as the antiviral immune response, immune cell proliferation and differentiation. In the present study, the involvement of a previously identified IRF8 homologue (CgIRF8) in regulating haemocyte proliferation of oyster were further investigated. CgIRF8 mRNA transcripts were detectable in all the stages of C. gigas larvae with the highest level in D-veliger (1.76-fold of that in zygote, p < 0.05). Its mRNA transcripts were also detected in all the three haemocyte subpopulations of adult oysters with the highest expression in granulocytes (2.79-fold of that in agranulocytes, p < 0.01). After LPS stimulation, the mRNA transcripts of CgIRF8 in haemocytes significantly increased at 12 h and 48 h, which were 2.04-fold and 1.65-fold (p < 0.05) of that in control group, respectively. Meanwhile, the abundance of CgIRF8 protein in the haemocytes increased significantly at 12 h after LPS stimulation (1.71-fold of that in seawater, p < 0.05). The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgIRF8 protein in haemocytes. After the expression of CgIRF8 was inhibited by the injection of CgIRF8 siRNA, the percentage of EdU positive haemocytes, the proportion of granulocytes, and the mRNA expression levels of CgGATA and CgSCL all declined significantly at 12 h after LPS stimulation, which was 0.64-fold (p < 0.05), 0.7-fold (p < 0.05), 0.31-fold and 0.54-fold (p < 0.001) of that in the NC group, respectively. While the expression level of cell proliferation-related protein CgCDK2, CgCDC6, CgCDC45 and CgPCNA were significantly increased (1.99-fold, and 2.41-fold, 3.76-fold and 4.79-fold compared to that in the NC group respectively, p < 0.001). Dual luciferase reporter assay demonstrated that CgIRF8 was able to activate the CgGATA promoter in HEK293T cells after transfection of CgGATA and CgIRF8. These results collectively indicated that CgIRF8 promoted haemocyte proliferation by regulating the expression of CgGATA and other related genes in the immune response of oyster.

Water stratification alters phytoplankton assemblages in scallop farming waters of the North Yellow Sea in China.

As evaluation indicators of the primary productivity, the phytoplankton biomass and community structure are of great significance to the fishery industry, which can be driven by ocean currents, nutrients and water stratification. In the present study, the characteristics of phytoplankton assemblages in different water layers of a typical Yesso scallop farming area in Zhangzi Island, the North Yellow Sea were investigated from March 2021 to January 2022. According to the vertical distribution of temperature, water stratification was observed from June to August (stratification period), and disappeared in March, October and the following January with vertical homogeneity (mixing period). 18S rRNA gene sequencing results revealed that Pyrrophyta was the most dominant phylum during the sampling period, with high gene proportions in the stratification (63.36%) and mixing periods (77.35%). The gene proportion of Bacillariophyta in the stratification period was 5.44%, which was significantly lower than that in the mixing period of 8.93% (p < 0.05). Moreover, Pseudo-nitzschia, a toxin-producing taxon affiliated with Bacillariophyta, exhibited a significantly higher proportion in the stratification period than in the mixing period. During the stratification period, a number of toxin-producing taxa such as Pseudo-nitzschia and Karlodinium were enriched in the bottom layer, which was 1.29-fold and 1.37-fold of that in the surface layer, respectively. Redundancy analysis showed that phosphate and water temperature were major environmental factors driving the vertical distribution of phytoplankton assemblages. The phosphate (0.11 µM) and silicate (2.09 µM) concentrations in the surface layer approached the minimum threshold for phytoplankton growth, and the stoichiometric limitation of phosphate was detected in the surface and middle layers. Collectively, these results indicated that the decreased proportion ratio of Bacillariophyta to Pyrrophyta and unfavorable community composition of Bacillariophyta for scallops were observed during summer, which might result from the phosphate limitation driven by water stratification. The results will further our understanding of the dynamics of phytoplankton communities under the background of intensifying ocean stratification and provide ecological guidance for mollusc mariculture.

A DM9-containing protein from crab Eriocheir sinensis functions as a novel multipotent pattern recognition receptor.

DM9-containing protein in invertebrates functions as pattern recognition receptor (PRR) to play significant roles in innate immunity. In the present study, a novel DM9-containg protein (defined as EsDM9CP-1) was identified from the Chinese mitten crab Eriocheir sinensis. EsDM9CP-1 is composed of 330 amino acids containing a Methyltransf_FA domain and two tandem DM9 repeats. The deduced amino acid sequence of EsDM9CP-1 shared low similarity with the previously identified DM9CPs from other species, and it was closely clustered with Platyhelminthes DM9CPs and then assigned into the branch of invertebrate DM9CPs in the unrooted phylogenetic tree. The mRNA transcripts of EsDM9CP-1 were highly expressed in haemocytes, gill, and heart. After Aeromonas hydrophila stimulation, the expression levels of EsDM9CP-1 mRNA in haemocytes increased significantly at 3 h (3.88-fold, p < 0.05) and 6 h (2.71-fold, p < 0.05), compared with that of PBS group, respectively. EsDM9CP-1 protein was mainly distributed in the cytoplasm and membrane of haemocytes. The recombinant EsDM9CP-1 protein (rEsDM9CP-1) exhibited binding affinity to MAN, PGN, LPS and Poly (I:C), and also to Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli, A. hydrophila and Vibrio splendidus) and fungi (Pichia pastoris and Metschnikowia bicuspidata) in a Ca2+-dependent manner. It was able to agglutinate A. hydrophila, S. aureus, M. luteus, M. bicuspidata and P. pastoris, and inhibit the growth of A. hydrophila and M. bicuspidate. These results suggested that EsDM9CP-1 in crab not only functioned as a PRR, but also agglutinated and inhibited the growth of microbes.

The Modification of H3K4me3 Enhanced the Expression of CgTLR3 in Hemocytes to Increase CgIL17-1 Production in the Immune Priming of Crassostrea gigas.

Increasing evidence confirms that histone modification plays a critical role in preserving long-term immunological memory. Immune priming is a novel form of immunological memory recently verified in invertebrates. Toll-like receptor (TLR) signaling and cytokines have been reported to be involved in the immune priming of the Pacific oyster Crassostrea gigas. In the present study, the expression of Toll-like receptor 3 (CgTLR3), myeloid differentiation factor 88-2 (CgMyd88-2) and interleukin 17-1 (CgIL17-1) was found to be elevated in the hemocytes of C. gigas at 6 h after the secondary stimulation with Vibrio splendidus, which was significantly higher than that at 6 h after the primary stimulation (p < 0.05). A significant increase in histone H3 lysine 4 trimethylation (H3K4me3) enrichment was detected in the promoter region of the CgTLR3 gene at 7 d after the primary stimulation with inactivated V. splendidus (p < 0.05). After the treatment with a histone methyltransferase inhibitor (5'-methylthioadenosine, MTA), the level of H3K4me3 at the promoter of the CgTLR3 gene decreased significantly at 7 d after the primary stimulation with inactivated V. splendidus (p < 0.05), and the expression of CgTLR3, CgMyD88-2 and CgIL17-1 was significantly repressed at 6 h after the secondary stimulation with V. splendidus (p < 0.05). Conversely, the treatment with monomethyl fumarate (MEF, an inhibitor of histone demethylases) resulted in a significant increase in H3K4me3 enrichment levels at the CgTLR3 promoter at 7 d after the primary stimulation (p < 0.05), and the expression of CgTLR3, CgMyD88-2 and CgIL17-1 was observed to increase significantly at 6 h after the secondary stimulation (p < 0.05). These results suggested that H3K4me3 regulated MyD88-dependent TLR signaling in the hemocytes of C. gigas, which defined the role of histone modifications in invertebrate immune priming.

Whole genome sequencing of a novel sea anemone (Actinostola sp.) from a deep-sea hydrothermal vent.

Deep-sea hydrothermal vents are usually considered as extreme environments with high pressure, high temperature, scarce food, and chemical toxicity, while many local inhabitants have evolved special adaptive mechanisms for residence in this representative ecosystem. In this study, we constructed a high-quality genome assembly for a novel deep-sea anemone species (Actinostola sp.) that was resident at a depth of 2,971 m in an Edmond vent along the central Indian Ocean ridge, with a total size of 424.3 Mb and a scaffold N50 of 383 kb. The assembled genome contained 265 Mb of repetitive sequences and 20,812 protein-coding genes. Taken together, our reference genome provides a valuable genetic resource for exploring the evolution and adaptive clues of this deep-sea anemone.

PyLKB1 regulates glucose transport via activating PyAMPKα in Yesso Scallop Patinopecten yessoensis under high temperature stress.

Liver kinase B1 (LKB1) is a classical serine/threonine protein kinase and plays an important role in maintaining energy homeostasis through phosphorylate AMP-activated protein kinase α subunit (AMPKα). In this study, a homologous molecule of LKB1 with a typical serine/threonine kinase domain and two nuclear localization sequences (NLSs) was identified in Yesso Scallop Patinopecten yessoensis (PyLKB1). The mRNA transcripts of PyLKB1 were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in hepatopancreas. PyLKB1 was mainly located in cytoplasm and nucleus of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyLKB1, PyAMPKα, and PyGLUT1 in hepatopancreas, the phosphorylation level of PyAMPKα at Thr170 in hepatopancreas, the positive fluorescence signals of PyLKB1 in haemocytes, glucose analogue 2-NBDG content in haemocytes, and glucose content in hepatopancreas, haemocytes and serum all increased significantly (p < 0.05) compared to blank group (15 °C). However, there was no significant difference at the protein level of PyLKB1 and PyAMPKα. After PyLKB1 was knockdown by siRNA, the mRNA expression level of PyGLUT1, and the glucose content in hepatopancreas and serum were significantly down-regulated (p < 0.05) compared with the negative control group receiving an injection of siRNA-NC. However, there were no significant difference in PyGLUT1 expression, glucose content and glucose analogue 2-NBDG content in haemocytes. These results collectively suggested that PyLKB1-PyAMPKα pathway was activated to promote glucose transport by regulating PyGLUT1 in response to high temperature stress. These results would be helpful for understanding the function of PyLKB1-PyAMPKα pathway in regulating glucose metabolism and maintaining energy homeostasis under high temperature stress in scallops.

Unveiling the functional diversity of ionotropic glutamate receptors in the Pacific oyster (Crassostrea gigas) by systematic studies.

Ionotropic glutamate receptors (iGluRs), pivotal in mediating excitatory neurosignals within the central nervous system, are instrumental in environmental stress responses. In this investigation, 12 iGluRs identified in the Pacific oyster are herein designated as CgiGluRs, and further categorized into three distinct subfamilies based on their transmembrane domains. Cross-species evolutionary analysis unveiled a high degree of conservation in the sequence and structural attributes of these CgiGluRs. These receptors are ubiquitously distributed across various tissues, with pronounced expression in the oyster's mantle, labial palps, and gills, underlining their integral role in the oyster's environmental sensing mechanisms. Post the D-shaped larval stage, a marked upward trend in CgiGluRs expression was observed, denoting their critical involvement in oyster development beyond this phase. Exposure to five metals-cadmium (Cd), copper (Cu), zinc (Zn), mercury (Hg), and lead (Pb)-elicited a significant upregulation of CgGRIA4 expression, indicating a robust response to metal stress. A KEGG enrichment analysis on 142 genes, exhibiting parallel expression trends with CgGRIA4 under metal stress, suggests that CgGRIA4 could augment excitatory signal transmission by activating glutamatergic and dopaminergic synapses, thereby contributing to the metal stress response in the oyster. This inquiry not only bolsters our comprehension of the iGluRs gene family in metal stress response but also paves the way for future exploration of its cardinal role in cellular signaling and environmental adaptability.